Technology Platforms

Conventional HEK293 AAV Production

HEK293 AAV Production leverages the well-characterized human embryonic kidney HEK293 cells for the generation of high-quality Adeno-Associated Virus (AAV) vectors. We use a three-plasmid transfection system, where one plasmid carries the AAV replication/capsid (rep/cap) genes, a second plasmid codes for the adenovirus helper genes, and the third encodes the gene of interest (GOI) flanked by two AAV inverted terminal repeats (ITRs). This method ensures:

• High Yield: Optimized for robust AAV production, generating up to 1E+15 vector genomes (vg) per batch.
• Consistency: Reproducible results across multiple batches, crucial for both research and clinical applications.
• Purity: High-level vector purity (low amounts of empty capsids, cell debris and free DNA) achieved through established purification protocols.
• Scalability: Adaptable from small-scale production to large-scale manufacturing.

Ideal for academic researchers and biopharmaceutical companies seeking reliable, scalable, and cost-effective AAV vector production for gene therapy applications.

Suspension cells are transfected with three plasmids: one that encodes adenovirus helper genes (E2A, E4, VA), one that carries the AAV replication/capsid (Rep/Cap) genes and one that encodes a gene of interest (GOI). This system yields high quality rAAV vectors.

This system produces AAV using Spodoptera frugiperda (Sf9) insect cells and recombinant baculoviruses (rBV). Two features enable this system to produce Adeno-Associated Virus (AAV) titers of up to 1E+17 vector genomes (vg)/batch: A-B. An artificial intron, containing an insect (polh) promoter, is inserted between nucleotide 850 and 851 of the AAV replication (Rep) genes to enable the expression of both Rep78 and Rep52, while making the baculovirus more stable. C. The expression levels of Rep78 and Rep52 in Sf9 cells transfected with the Bacmid or infected with rBV carrying the artificial polh promoter-containing intron embedded in the Rep genes (lanes 1 & 2) are significantly higher than those reported from plasmid transfected HEK293 cells. D-E. The same artificial intron, containing the insect polh promoter is inserted between nucleotide 2227 and 2228 of the AAV capsid (Cap) genes to enable higher expression of VP1 (one of three AAV capsid proteins), while maintaining high expression levels of the other capsid proteins (VP2 and VP3). This results in highly infectious AAV particles that are lacking in first generation baculovirus system. F. Higher levels of VP1 are produced and packaged into AAV virions using the rBVs carrying the artificial polh promoter intron-containing Rep and Cap coding sequences (lane 2) than by using those carrying artificial intron-less Rep and start codon–mutated Cap coding sequences (lane 1).

BAC-to-AAV Sf9 High Titer AAV Production System

Precision AAV Capsid Engineering (PACE)

PACE is an innovative platform technology designed for AAV capsid modifications that allows specific targeting of cell types of interest. Key features include:

• Precision Design: Utilizes existing databases to screen for existing targeting moieties that can be used to modify capsids to achieve structural stability, enhanced tropism, improved efficacy, and immune evasion for maximal potency.
• Customization: Tailors AAV vectors to specific cell types or tissues, improving delivery and therapeutic efficacy, while minimizing off-target expression.
• Rapid Prototyping: Accelerates the development cycle from design to functional capsid variant selection and testing.
• Scalability: Adaptable for both small-scale research and large-scale therapeutic applications.

Ideal for researchers and developers aiming to optimize gene delivery vectors for precision medicine and novel therapeutic applications.

This technology modifies AAV capsids to enable specific targeting of cell types of interest (in this example, a T cell-targeting moiety, TCeT). A. Healthy human Peripheral Blood Mononuclear Cells (PBMCs) were either not transduced (left panel), transduced with non-targeted wild-type (wt) AAV expressing eGFP (middle panel) or transduced with TCeT-modified AAV expressing eGFP targeting T cells (right panel). Transduction efficiency was assessed by flow cytometry. B. Fold transduction difference of T cells transduced with TCeT-modified-AAV vs. Wt-AAV from 5 healthy donors. The PACE technology can be used to target additional cell types that have lineage or tumor-specific markers.

A. Wildtype (wt) baculovirus (BV) contains only the wt genome. B. Recombinant BV (rBV) contains the wt genome and a gene of interest (GOI). C. A different rBV contains the wt genome, a GOI and a drug resistance cassette. D. As rBV containing the wt genome and the GOI are passaged in Spodoptera frugiperda (Sf9) cells every 3-4 days for 10 passages, the rBV loses the GOI by passage 10. E. As rBV containing the wt genome, the GOI and a drug resistance cassette are passaged in Sf9 cells every 3-4 days for 10 passages in the presence of a selection drug, the rBV maintains the GOI for up to 10 passages.

High-Fidelity (Hi-Fi) Recombinant Baculovirus (rBV) System

Technology for Toxic Gene Packaging in AAV Vectors

Toxic Gene Packaging in AAV: Historically, it has not been possible to package toxic genes in AAV using insect or mammalian cells because the cells synthesize the toxin and die. Our novel patented technology overcomes this limitation by:

• Intron Splicing: Due to existing differences in intron splicing mechanisms between insect and mammalian cells, it is possible to package an encoded toxic gene because it is not expressed during vector manufacturing and does not have a negative impact on recombinant baculovirus (rBV) and AAV production.
• High Yield: Enables the production of AAV vectors with toxic genes at yields that are comparable to vectors without toxic genes, thus facilitating research and development of gene therapies for conditions that would benefit from the expression of a toxic gene.
• Therapeutic Potential: Broadens the scope of AAV-mediated gene therapies to include applications involving cytotoxic or apoptosis-inducing genes, which was previously impossible due to vector production constraints.

This platform technology opens new avenues for gene therapy, particularly in cancer treatments and other conditions that require controlled cell death.

A. The toxic gene packaging system works by turning off toxin expression in Sf9 cells during AAV manufacturing using a mammalian intron insertion. Sf9 cells cannot splice out mammalian introns, so no toxin is produced. B. The toxin expression system is turned on in mammalian cancer cells upon AAV entry following intron splicing. C. Pancreatic cells (PANC-1) or lung fibroblasts (WI38) were infected with AAV carrying diphtheria toxin A (AAV-DTA) or GFP (AAV-GFP) under the control of a tumor-specific promoter. The tumor-specific promoter is active in PANC-1 but not in WI38 cells, as evidenced by the destruction of the PANC-1 cell monolayer, while leaving an intact WI38 monolayer. D. Dose response curves showing the fold change in cell growth (expressed in relative light units, RLU) with increasing concentrations of AAV-DTA or AAV-GFP. PANC-1 cell growth was inhibited by AAV-DTA, while WI38 growth was not.

Suspension cells are transfected with three plasmids: one that encodes an envelope and the REV gene (VSV-G/REV), one that encodes packaging and replication/DNA integration proteins (Gag/Pol) and one that encodes a gene of interest (GOI) in the Self-Inactivating Long Terminal Repeats (SIN-LTRs). This transfection system produces lentivirus with very high titers.

Lentivirus Production

AdONE Adenovirus Production Platform

AdONE Platform: Our AdONE platform revolutionizes adenovirus generation using a one-day process that features:

• Rapid Insertion: Enables the fast insertion of your gene of interest (GOI) into the adenovirus genome.
• Efficient Transfection: Directly transfects the engineered adenovirus genome into HEK293 cells on the same day for immediate adenovirus production.
• High Yield: Delivers high-titer adenovirus in a significantly reduced timeframe.
• Simplicity: Streamlines the adenovirus production workflow by minimizing labor and manufacturing time.

This technology accelerates the research or therapeutic development of cell and gene therapies by offering high-titer, quality adenovirus production, while drastically reducing turn-around time.

AdONE is our platform for a one-day process of gene of interest (GOI) insertion into the adenoviral genome and transfection of HEK293 cells for the purposes of subsequent recombinant adenovirus generation. HEK293 cells were transfected and recombinant adenovirus expressing EGFP was harvested after 5 days. The harvested virus was used to infect HEK293 cells for expansion and images of the cells were taken using fluorescent microscopy of a plaque (white ellipse) which formed 7 days after infection. A. Brightfield image of HEK293. B. EGFP image of HEK293.